β1-3- N-Acetylglucosaminyltransferase in Human Leukocytes: Properties and Role in Regulating Neolacto Glycosphingolipid Biosynthesis

Abstract

There are well-established differences in the expression of neolacto neutral glycosphingolipids among human leukocyte subclasses. Mature myeloid cells express several types of these compounds, whereas mature lymphoid cells are deficient in such compounds. The biochemical basis for this is unknown. Therefore, enzyme studies were done to determine whether different classes of leukocytes (represented by cell lines) contained β1-3- N-acetylglucosaminyltransferase activity (EC 2.4.1.149, β1-3GlcNAcT(i)). This enzyme participates in the synthesis of Type 2 chains in glycosphingolipids by catalyzing the following two reactions: (i) Galβ1-4Glcβ1-1Cer (lactosylceramide, LacCer) + UDP-GlcNAc → GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (lactotriaosylceramide) and (ii) Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (neolactotetraosylceramide) + UDP-GlcNAc → GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer. The first reaction may be the key step in the biosynthetic pathway of neolacto structures in human leukocytes. Therefore, extracts from several cell lines representative of both myeloid and lymphoid lineages, at varied stages of maturity, were assayed with LacCer for the presence of β1-3GlcNAcT(i) activity. Our results indicate that myeloid cells contain this initiating β1-3GlcNAcT(i) activity, whereas lymphoid cells do not. This is consistent with our thin-layer chromatography immunostain results which show that all myeloid cell lines express neutral neolacto glycosphingolipids and lymphoid cells do not. Our findings suggest that the presence of the initiating activity only in myeloid cells is a regulatory factor in the expression of neutral neolacto glycosphingolipids in human leukocytes. We also tested both myeloid and lymphoid cell lines for the presence of elongating β1-3GlcNAcT(i) activity (reaction (ii) above) by using neolactotetraosylceramide as an acceptor. Our results show that an elongating activity is expressed by all myeloid and lymphoid cell lines tested. Initiating (myeloid) and elongating (myeloid and lymphoid) activities were distinguished by several characteristics: metal ion activation, pH optimum, and kinetic constants. In conclusion, our results indicate the presence of two β1-3GlcNAcT(i) activities in human leukocytes: one that catalyzes the initial reaction and is found only in the myeloid lineage and one that catalyzes the elongating reaction and is found in both myeloid and lymphoid cells.